The present invention provides a novel sialidase and DNA coding for it. More precisely, the present invention provides sialidase that localizes in plasma membrane, and specifically hydrolyzes gangliosides, and DNA coding for it.
The sialidase of the present invention and the DNA coding for it are expected to be utilized as a reagent used for saccharide chain studies and a medicament used for gene diagnosis and gene therapy.
Sialidase is a glycohydrolytic enzyme present in living bodies, which eliminates a sialic acid residue from a non-reducing terminal of saccharide chains of glycoproteins or glycolipids. It has been known that, when sialic acid is removed from saccharide chain molecules, not only the degradation of these molecules begins, but also molecular conformation and many of important cell functions such as recognition mechanism by receptors, cell adhesion, and immunomechanism may be changed. It has also become clear that sialidase exhibits rapid activity change in connection with proliferation and canceration of cells, and it is involved in the metastatic ability of cancer cells. However, there is little knowledge about how sialic acid is eliminated in vivo. This is because the studies of mammalian sialidases on the molecular level are behindhand, and hence there are many unknown points concerning their structures and expression mechanism.
Because mammalian sialidase exhibits only low activity, and is extremely unstable, isolation and purification of the enzyme have been difficult. Sialidase has been often considered for a long time to be one of the mere lysosomal enzymes involved in the dissimilation and degradation. Under such a situation, we isolated and purified the enzyme by using mainly rat tissues as the source of the enzyme, and found that there were four types of sialidases which differ from sialidases of bacteria, viruses, protozoa and the like in their natures (Miyagi, T. and Tsuiki, S., Eur. J. Biochem. 141, 75-81, 1984; Miyagi, T. et al., J. Biochem. 107, 787-793, 1990; Miyagi, T. and Tsuiki, S., J. Biol. Chem. 260, 6710-6716, 1985). These enzymes each localize in lysosomal matrix, lysosome membrane, plasma membrane (cell surface membrane), and cytoplasm within a cell, respectively, and they are different from each other not only in enzymological characteristics such as substrate specificity, but also in immunological properties. Among those sialidases, the sialidase localized in cytoplasm can be obtained as a homogenous purified product from rat skeletal muscles. Its cDNA cloning has been succeeded for the first time in the world as for animal sialidases, and its primary structure has been determined (Miyagi T. et al., J. Biol. Chem., 268, 26435-26440, 1993). Its genomic structure analysis has also been done, and as for its function, it has been elucidated that the enzyme participates in the differentiation and the growth of skeletal muscle cells by using the cDNA as a probe. These studies can be considered a part of pioneer researches in sialidase studies in the world.
By the previous studies, it has become clear that there is possibility that the sialidase localized in plasma membrane exhibits activity elevation upon proliferation and canceration of cells, and it is also deeply concerned with the differentiation of nerve cells and the signal transduction of cells. To date, however, it has not been understood at all about the structure of this enzyme, the mechanism causing the activity change and the like. In order to answer these questions, what many researchers in this field have long been desired is cloning of its cDNA. For example, if the mechanism of cancerous change due to this enzyme could be elucidated, it would be possible to utilize the results in diagnosis and therapy of cancers. Further, because gangliosides exist in surface membranes of many cells and participate in important cell functions such as cell adhesion and informational communication, and they are also main cerebral components, the sialidase utilizing them as a specific substrate is estimated to be involved in certain important cranial nerve functions.
The present invention has been accomplished in view of the aforementioned present condition. An object of the present invention is to provide the sialidase localized in plasma membrane and DNA that codes for it.
The inventors of the present invention earnestly conducted studies in order to achieve the aforementioned object, and as a result, succeeded in isolating the sialidase localized in plasma membrane and cloning of cDNA coding for it. Furthermore, they found that the aforementioned sialidase was unique in that it substantially specifically hydrolyzed gangliosides (glycolipids containing sialic acid), which are substrates that similarly localize mainly in plasma membrane, and it was completely different from other mammalian sialidases and microbial sialidases in enzymatic substrate specificity. Thus, they accomplished the present invention.
That is, the present invention provides a protein defined in the following (A) or (B):
(A) a protein which has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or
(B) a protein which has the amino acid sequence including substitution, deletion, insertion, or transition of one or several amino acid residues in SEQ ID NO: 2 or SEQ ID NO: 4, and exhibits activity to eliminate a sialic acid residue from a non-reducing terminal of ganglioside.
The present invention also provides DNA coding for the protein defined in the above (A) or (B). Specifically, such DNA may be DNA which has the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
A sialidase which has the aforementioned characteristics will be referred to as the xe2x80x9csialidase of the present inventionxe2x80x9d hereinafter, and DNA coding for it will be referred to as the xe2x80x9cDNA of the present inventionxe2x80x9d hereinafter.
Hereafter, the present invention will be explained in detail.
 less than 1 greater than  Sialidase of the Present Invention
The sialidase of the present invention is a protein which has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Moreover, the sialidase of the present invention include a protein which has the amino acid sequence including substitution, deletion, insertion, or transition of one or several amino acid residues in SEQ ID NO: 2 or SEQ ID NO: 4, so long as it exhibits activity for eliminating a sialic acid residue from a non-reducing terminal of ganglioside.
Among the aforementioned sialidases, the sialidase that has the amino acid sequence of SEQ ID NO: 2 has the following physicochemical properties.
(1) Activity
It eliminates sialic acid residues from a non-reducing terminal of ganglioside.
(2) Substrate Specificity
It acts on gangliosides, but not act on glycoproteins and oligosaccharides. Specifically, it acts on GD3-ganglioside, GD1a-ganglioside, GM3-ganglioside, and synthetic gangliosides (GSC-17(xcex12-3) and GSC-61(xcex12-6)), but it does not substantially act on GM2-ganglioside, GM1-ganglioside, orosomucoid, fetuin, glycophorin, ovine submaxillary gland mucin, and bovine submaxillary gland mucin. It weakly acts on xcex12-3 sialyllactose, and 4-MUNeuAc (4-methylumbelliferyl N-acetylneuraminic acid).
(3) Optimum pH
4.7 to 5.0
(4) Molecular Weight
About 65,000 as determined by sucrose density gradient centrifugation,
About 52,000 as determined by SDS-polyacrylamide gel electrophoresis under reducing condition
(5) Inhibition and Activation
A surface active agent is required for the activity. For example, it is highly active in the presence of 0.1 to 0.2% of Triton X-100.
It is strongly inhibited by heavy metal ions such as Cu2+, and 4-hydroxy mercury benzoate.
It is stabilized by dithiothreitol, Neu5Ac2en (2-deoxy-2,3-dehydro-N-acetylneuraminic acid), and glycerol. However, it is weakly inhibited by Neu5Ac2en.
Among the sialidases of the present invention, the sialidase which has the aforementioned characteristics is an enzyme derived from bovine, whereas the sialidase which has the amino acid sequence of SEQ ID NO: 4 in one derived from human. These exhibit 82% homology in their amino acid sequences, and they have a transmembrane domain, glycosylation site, and Asp-box, which is a consensus sequence of sialidase, at the same locations. Therefore, the enzyme derived from human is considered to have the same physicochemical properties as the enzyme derived from bovine.
The sialidase of the present invention can be obtained from a bovine brain, for example, as follows. All of the following procedures are preferably carried out at a low temperature.
A bovine brain is homogenized in a buffer, and centrifuged at 1000xc3x97g for 10 minute, and the supernatant is further centrifuged at 30,000xc3x97g for 1 hour. After the centrifugation, the precipitate fraction is suspended in a buffer, added 5% deoxycholic acid, sufficiently homogenized, and centrifuged at 100,000xc3x97g for one hour to obtain a soluble fraction as a supernatant. The buffer preferably contains an inhibitor for proteases, dithiothreitol, surface active agent and the like.
The above soluble fraction is applied to a DEAE-cellulose column and, after the column is washed, eluted with a buffer containing 0.2 M NaCl for fractionation. A fraction exhibiting the sialidase activity is dialyzed against a buffer, then applied on Octyl-Sepharose, and separated by elution with a linear gradient of 0.1-0.4% Triton X-100.
Then, an active fraction is applied to Heparin-Sepharose (Pharmacia), washed with a buffer containing 0.25 M NaCl, and eluted with a 0.2-1 M NaCl linear gradient to concentrate the active fraction. The above concentrated enzyme solution is loaded on Sephacryl S-200 (Pharmacia), and separated by elution with a buffer containing 0.02 mM NeuAc2en (2-deoxy-2,3-dehydro-N-acetylneuraminic acid).
The obtained active fraction is diluted to have a Triton X-100 concentration of 0.02%, added to RCA-lectin agarose (Pharmacia), washed with a buffer containing 0.02% Triton X-100, and eluted with a buffer containing 0.2 M lactose. This active fraction is loaded on a MonoQ (Pharmacia) column, and eluted with a 0-0.5 M NaCl linear gradient.
The active fraction is loaded on an activated thiol Sepharose (Pharmacia) column, washed with a 0.15 M NaCl buffer containing 10% glycerol, and then with 0.5 M NaCl buffer containing 10% glycerol, and eluted with 0.05 M NaCl buffer containing 0.05 M dithiothreitol. The active fraction is concentrated in a MonoQ column.
The above concentrate is loaded on an affinity column utilizing a synthetic ganglioside GM3 [GSC-211, NeuAc-Gal-Glc-O(CH2)8NH2] as a ligand (Hasegawa A. et al. J. Carbohydr. Chem., 9, 201-214, 1990), and separated by elution with a 0-0.5 M NaCl gradient. The affinity column can be obtained by allowing GSC-211 to couple with ECH-Sepharose (Pharmacia) in the presence of N-ethyl-Nxe2x80x2-(3xe2x80x2-dimethyl-aminopropyl)carbodiimide hydrochloride.
The sialidase enzyme is purified as described above as a protein having a molecular weight of 52 kD as determined by SDS-polyacrylamide gel electrophoresis.
Further, since DNA coding for the sialidase of the present invention has been obtained, the sialidase of the present invention can also be obtained by expressing the DNA in a suitable host-vector system. As for the host-vector system, a cultured cell can be used as a host, and a vector suitable for this host can be used. Materials and methods therefore may be those usually used for the production of heterogenous proteins utilizing genetic recombination techniques. When DNA coding for the sialidase of the present invention is ligated to a vector, a vector containing sequences required for regulation of gene expression such as promoter and terminator that can be expressed in the host as required may be used.
 less than 2 greater than  DNA of the Present Invention
Because the amino acid sequence of the protein encoded by the DNA of the present invention has been elucidated, the DNA of the present invention can be cloned based on the amino acid sequence. In the examples mentioned below, a partial amino acid sequence of the sialidase of the present invention is determined, oligonucleotide primers are synthesized based on the partial amino acid sequence, and the DNA of the present invention is obtained from a bovine brain cDNA library by PCR (polymerase chain reaction) using the oligonucleotides primers.
Although the sequence of the DNA of the present invention is not particularly limited so long as it codes for the amino acid sequence of SEQ ID NO: 2 or 4, the nucleotide sequences of SEQ ID NO: 1 and 3 can be specifically mentioned. Further, existence of sialidases having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 including substitution, deletion, insertion, or transition of one or several amino acid residues, and genes coding for them is expected due to difference of animal species, individuals or varieties. Such DNA coding for the substantially same protein as the sialidase of the present invention also falls within the scope of the DNA of the present invention. Such DNA can also be obtained from a cell harboring it by hybridizing it with the nucleotide sequence of SEQ ID NO: 1 or 3 or a part thereof under a stringent condition, and isolating DNA coding for a protein which has sialidase activity. DNA coding for a sialidase having such a mutation as mentioned above may also be obtained by, for example, site-specific mutagenesis or mutagenic treatment.
The term xe2x80x9cone or severalxe2x80x9d amino acid residues means 1-80, preferably 1-30, more preferably 1-5 amino acid residues.
 less than 3 greater than  Pregressive Applications of the Sialidase of the Present Invention and DNA Coding for it
(1) Because the sialidase of the present invention exhibits substrate specificity unique in that it substantially specifically hydrolyze gangliosides, a recombinant having the enzyme or DNA coding for the enzyme has much possibility for use as a reagent for saccharide chain studies.
(2) As one of the means for normalizing abnormality of this enzyme observed in cancer cells, for example, antisense therapy, which is a kind of gene therapy, will be expected in future. The gene structure clarified by the present invention is the important information for it. Moreover, if the expression mechanism of this enzyme becomes clear by genome structure analysis utilizing the cDNA as a probe, it will also become possible to normalize the abnormality of the expression of this enzyme in cancer and the like.
(3) Because of two reasons, i.e., the characteristic that the enzyme specifically decomposes gangliosides that are main components of brain, and its involvement in differentiation of nerve cells, the abnormalities of this enzyme may be found in certain brain diseases. In such a case, the information about the gene may be much utilized for development of gene therapy and medicaments.